Method for production of recombinant human iduronate 2-sulfatase

ABSTRACT

Disclosed is a method for production of recombinant human iduronate 2-sulfatase (rhI2S) (the 26th to 550th amino acids of SEQ ID NO: 10) in a large scale, with a high purity, and mannose 6-phosphate residues. The method comprises the steps of (a) culturing rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10)-producing mammalian cells in a serum-free medium, (b) collecting culture supernatant, (c) subjecting the culture supernatant to cation-exchange column chromatography, (d) to dye affinity column chromatography, (e) to anion-exchange column chromatography, and (f) to a column chromatography employing as solid phase a material having affinity for phosphate group, and (g) to gel filtration column chromatography, in the order.

The instant application is a national stage application ofPCT/JP2012/000365, filed Jan. 23, 2012.

TECHNICAL FIELD

The present invention relates to a method for production of recombinanthuman iduronate 2-sulfatase (rhI2S) (the 26th to 550th amino acids ofSEQ ID NO: 10). More specifically the present invention relates to aprocess for production of rhI2S (the 26th to 550th amino acids of SEQ IDNO: 10) by culturing rhI2S (the 26th to 550th amino acids of SEQ ID NO:10) producing mammalian cells in a serum free medium, as well as to aprocess for purification of rhI2S (the 26th to 550th amino acids of SEQID NO: 10) obtained in the culture supernatant through columnchromatography, in high yield and to such a high purity as permitsdirect use of the purified protein as a medical drug. The presentinvention further relates to glycosylated rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) which is characterized as having an ability toundergo highly efficient cellular uptake, strong affinity for themannose-6-phosphate (M6P) receptor, and as having a linkedoligosaccharide chain with high mannose-6-phosphate content.

BACKGROUND ART

Iduronate 2-sulfatase (I2S) is a lysosomal enzyme having an activity tohydrolyze a sulfate ester bond in a glycosaminoglycan molecule such asheparan sulfate and dermatan sulfate. Patients of Hunter's syndromegenetically lack I2S activity. Lack of this enzyme causes abnormalmetabolism of heparan sulfate and dermatan sulfate, which then leads toaccumulation of fragments of the latter molecules in tissues such as theliver and kidney, and also to excretion of heparan sulfate and dermatansulfate in urine. These abnormalities then cause diverse symptoms inpatients suffering Hunter's syndrome, including skeletal deformities andsevere mental retardation.

The fact that the patients suffering Hunter's syndrome show scarce I2Sactivity has already been known since 1970's, and an abnormality of I2Sgene was expected to be the cause of this disease. In 1999, human geneencoding I2S was isolated and confirmed to be the responsible gene forthis disease (see LPL 1).

Based on the fact mentioned above, I2S replacement has been attemptedsince 1970's to improve the clinical conditions of the patients,including transplantation of normal macrophage (see LPL 2) and infusionof normal serum (see LPL 3) to supplement I2S activity in the patients.The results of those attempts showed that the concentration of heparansulfate in patients' urine decreased by the supplementation of I2Sactivity, suggesting that I2S replacement therapy should have a clinicalefficacy against Hunter's syndrome, though improvement in clinicalsymptoms was not confirmed. Practical application of the I2S replacementtherapy, however, has been extremely limited due to the lack of supplyof this enzyme.

Isolation of the gene encoding I2S in 1999 made it possible to produceI2S in a large scale using recombinant technology, and to use thisenzyme as a medicament for the enzyme replacement therapy for Hunter'ssyndrome (see PTL 1). However a method has not been reported so far forproviding I2S with such a high purity as is sufficient for the enzyme tobe directly used as a medical drug.

CITATION LIST Patent Literature

[PTL 1]

-   U.S. Pat. No. 5,932,211

Non Patent Literature

[NPL 1]

-   Wilson P J et al., Proc Natl Acad Sci USA. 87: 8531-5, 1990.    [NPL 2]-   Dean M F et al., J Clin Invest. 63: 138-45, 1979.    [NPL 3]-   Brown F R 3^(rd) et al., Am J Med Genet. 13 309-18, 1982.

SUMMARY OF INVENTION Technical Problem

Against the above background, it is an objective of the presentinvention to provide a method for production of recombinant human I2S(rhI2S) (the 26th to 550th amino acids of SEQ ID NO: 10) starting withculturing of rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10)producing mammalian cells in a serum free medium.

It is another objective of the present invention to provide a processfor purification of rhI2S (the 26th to 550th amino acids of SEQ ID NO:10) obtained in the culture supernatant, through column chromatography,in high yield and to such a high purity as permits direct use of thepurified protein as a medical drug.

Solution to Problem

The present inventors found that rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) contained in the supernatant of a culture of rhI2S (the26th to 550th amino acids of SEQ ID NO: 10)-producing cells in aserum-free medium, can be purified to a very high purity, and in a veryhigh yield as well, by subjecting the rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) to a purification process consisting of acombination of cation-exchange column chromatography, dye-affinitycolumn chromatography, anion-exchange column chromatography,chromatography using a column having affinity for phosphate group, andgel filtration column chromatography. The present invention wascompleted through further studies based on the finding.

Thus, the present invention provides what follows:

1. A method for production of recombinant human iduronate 2-sulfatase(rhI2S) (the 26th to 550th amino acids of SEQ ID NO: 10) comprising thesteps of:

(a) culturing rhI2S (the 26th to 550th amino acids of SEQ ID NO:10)-producing mammalian cells in a serum-free medium to let them secreterhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) in the medium,

(b) collecting culture supernatant by removing the cells from theculture that is obtained in step (a) above,

(c) subjecting the culture supernatant collected in step (b) above tocation-exchange column chromatography to collect rhI2S (the 26th to550th amino acids of SEQ ID NO: 10)-active fractions,

(d) subjecting the fractions collected in step (c) above to dye affinitycolumn chromatography to collect rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10)-active fractions,

(e) subjecting the fractions collected in step (d) above toanion-exchange column chromatography to collect rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10)-active fractions,

(f) subjecting the fractions collected in step (e) above to a columnchromatography employing as solid phase a material having affinity forphosphate group to collect rhI2S (the 26th to 550th amino acids of SEQID NO: 10)-active fractions, and

(g) subjecting the fractions collected in step (0 above to gelfiltration column chromatography to collect rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10)-active fractions, in the order.

2. The method according to (1) above, wherein the cation exchangeremployed in the cation-exchange column chromatography is a weak cationexchanger.

3. The method according to (2) above, wherein the weak cation exchangerhas a selectivity based on both hydrophobic interaction and hydrogenbond formation.

4. The method according to (2) or (3) above, wherein the weak cationexchanger has phenyl groups, amide bonds and carboxyl groups.

5. The method according to one of (1) to (4) above, wherein the dyeemployed in the dye affinity column chromatography is a blue triazinedye.

6. The method according to one of (1) to (5) above, wherein the materialhaving affinity for phosphate group is selected from the groupconsisting of fluoroapatite and hydroxyapatite.

7. The method according to (6) above, wherein the material havingaffinity to phosphate group is fluoroapatite.

8. The method according to one of (1) to (7) above, wherein themammalian cells are CHO cells transfected with an expression vectorwhich is designed to express rhI2S (the 26th to 550th amino acids of SEQID NO 10) under the regulation of EF-1(alpha) promoter.

9. A method for purification of rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) from contaminants, wherein the rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) has an oligosaccharide chain linkedthereto containing one or more mannose 6-phosphate residues, and whereinthe method comprises the steps of

(a) applying rhI2S (the 26th to 550th amino acids of SEQ ID NO 10) withcontaminants to a chromatography column which employs as solid phase amaterial having affinity for phosphate group,

(b) flowing a first mobile phase through the column to wash the columnwhile letting rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) beadsorbed by the column, and

(c) eluting rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) fromthe column by flowing a second mobile phase through the column, whereinthe second mobile phase contains a phosphate at higher concentrationthan the first mobile phase.

10. The method according to (9) above, wherein the material havingaffinity for phosphate group is selected from the group consisting offluoroapatite and hydroxyapatite.

11. The method according to (10) above, wherein the material havingaffinity for phosphate group is fluoroapatite.

12. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) having anoligosaccharide chain linked thereto which contains one or moremannose-6-phosphate residues, wherein the average number of themannose-6-phosphate residues per rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) molecule is in the range of 3.5 to 6.0.

13. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) according to(12) above having an oligosaccharide chain linked thereto which containsone or more mannose-6-phosphate residues, wherein the average number ofthe mannose-6-phosphate residues per rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) molecule is in the range of 3.6 to 5.5.

14. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) according to(12) above having an oligosaccharide chain linked thereto which containsone or more mannose-6-phosphate residues, wherein the average number ofthe mannose-6-phosphate residues per rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) molecule is in the range of 3.7 to 5.4.

15. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) according to(12) above having an oligosaccharide chain linked thereto which containsone or more mannose-6-phosphate residues, wherein the average number ofthe mannose-6-phosphate residues per rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) molecule is in the range of 4.0 and 6.0.

16. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) according to(12) above having an oligosaccharide chain linked thereto which containsone or more mannose-6-phosphate residues, wherein the average number ofthe mannose-6-phosphate residues per rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) molecule is in the range of 4.2 and 5.8.

17. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) according to(12) above having an oligosaccharide chain linked thereto which containsone or more mannose-6-phosphate residues, wherein the average number ofthe mannose-6-phosphate residues per rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) molecule is in the range of 4.5 and 5.4.

18. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) according toone of (12) to (17) above, wherein the average dissociation constantbetween the rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) andmannose-6-phosphate receptor is in the range of 7.5 to 20×10⁻¹⁰ mol/L.

19. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) according toone of (12) to (17) above, wherein the average dissociation constantbetween the rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) andmannose-6-phosphate receptor is in the range of 7.5 to 15×10⁻¹⁰ mol/L.

20. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) according toone of (12) to (17) above, wherein the average dissociation constantbetween the rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) andmannose-6-phosphate receptor is in the range of 4.5 to 20×10⁻¹⁰ mol/L.

21. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) according toone of (12) to (17) above, wherein the average dissociation constantbetween the rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) andmannose-6-phosphate receptor is in the range of 5.0 to 15×10⁻¹⁰ mol/L.

22. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) produced bythe method according to one of (1) to (11) above, wherein the rhI2S (the26th to 550th amino acids of SEQ ID NO: 10) has an oligosaccharide chainlinked thereto containing one or more mannose-6-phosphate residues, andwherein the average number of the mannose-6-phosphate residues per rhI2S(the 26th to 550th amino acids of SEQ ID NO: 10) molecule is in therange of 3.5 to 6.0.

23. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) produced bythe method according to one of (1) to (11) above, wherein the rhI2S (the26th to 550th amino acids of SEQ ID NO: 10) has an oligosaccharide chainlinked thereto containing one or more mannose-6-phosphate residues, andwherein the average number of the mannose-6-phosphate residues per rhI2S(the 26th to 550th amino acids of SEQ ID NO: 10) molecule is in therange of 3.6 to 5.5.

24. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) produced bythe method according to one of (1) to (11) above, wherein the rhI2S (the26th to 550th amino acids of SEQ ID NO: 10) has an oligosaccharide chainlinked thereto containing one or more mannose-6-phosphate residues, andwherein the average number of the mannose-6-phosphate residues per rhI2S(the 26th to 550th amino acids of SEQ ID NO: 10) molecule is in therange of 3.7 to 5.4.

25. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) produced bythe method according to one of (1) to (11) above, wherein the rhI2S (the26th to 550th amino acids of SEQ ID NO: 10) has an oligosaccharide chainlinked thereto containing one or more mannose-6-phosphate residues, andwherein the average number of the mannose-6-phosphate residues per rhI2S(the 26th to 550th amino acids of SEQ ID NO: 10) molecule is in therange of 4.0 to 6.0.

26. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) produced bythe method according to one of (1) to (11) above, wherein the rhI2S (the26th to 550th amino acids of SEQ ID NO: 10) has an oligosaccharide chainlinked thereto containing one or more mannose-6-phosphate residues, andwherein the average number of the mannose-6-phosphate residues per rhI2S(the 26th to 550th amino acids of SEQ ID NO: 10) molecule is in therange of 4.2 to 5.8.

27. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) produced bythe method according to one of (1) to (11) above, wherein the rhI2S (the26th to 550th amino acids of SEQ ID NO: 10) has an oligosaccharide chainlinked thereto containing one or more mannose-6-phosphate residues, andwherein the average number of the mannose-6-phosphate residues per rhI2S(the 26th to 550th amino acids of SEQ ID NO: 10) molecule is in therange of 4.5 to 5.4.

28. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) produced bythe method according to one of (1) to (11) above, wherein the averagedissociation constant between the rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) and mannose-6-phosphate receptor is in the range of7.5 to 20×10⁻¹° mol/L.

29. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) produced bythe method according to one of (1) to (11) above, wherein the averagedissociation constant between the rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) and mannose-6-phosphate receptor is in the range of7.5 to 15×10⁻¹° mol/L.

30. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) produced bythe method according to one of (1) to (11) above, wherein the averagedissociation constant between the rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) and mannose-6-phosphate receptor is in the range of4.5 to 20×10⁻¹° mol/L.

31. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) produced bythe method according to one of (1) to (11), wherein the averagedissociation constant between the rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) and mannose-6-phosphate receptor is in the range of5.0 to 15×10⁻¹⁰ mol/L.

32. RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) produced bythe method according to one of (1) to (11) above.

Advantageous Effects of Invention

As it has enabled to produce rhI2S (the 26th to 550th amino acids of SEQID NO: 10) starting with serum-free culturing of cells, the presentinvention provides rhI2S (the 26th to 550th amino acids of SEQ ID NO:10) which is free of any serum-derived contaminants including pathogenicagents such as viruses or prions. Therefore, the rhI2S (the 26th to550th amino acids of SEQ ID NO: 10) obtained according to the presentinvention can be administered into a human body as a safe medicamentsubstantially without any risks of exposure to such pathogenic agents.Further, as it has enabled purification of rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) substantially avoiding any use of organicsolvents, the present invention eliminates the risk of denaturation ofrhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) which otherwisemight be caused by the exposure to an organic solvent employed.Furthermore, the present invention is favorable to the environment, forthe waste fluid left after performing the purification process accordingto it does not contain an organic solvent, and in the economic sense aswell, for it requires no facility in which to treat organic solventswhich would otherwise be contained in the waste fluid.

Further the present invention makes it possible to selectively purifyrhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) having mannose6-phosphate residue in its oligosaccharide chain. To exert its enzymaticactivity after administration to a human body, rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) should be taken up by relevant cellsthrough mannose 6-phosphate receptors expressed on their surface.Therefore the efficacy of rhI2S (the 26th to 550th amino acids of SEQ IDNO: 10) as a medicament is dramatically increased through the selectivepurification of rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10)according to the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1-1 shows a first part of a schematic diagram illustrating themethod for construction of vector pE-neo/hGHpA(I2S).

FIG. 1-2 shows a second part of a schematic diagram illustrating themethod for construction of vector pE-neo/hGHpA(I2S).

FIG. 2 shows the growth curve of recombinant cells for expression ofrhI2S (the 26th to 550th amino acids of SEQ ID NO: 10).

FIG. 3 shows the pattern obtained by SDS-PAGE electrophoresis ofpurified rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10). Tolanes 1 to 3 was applied 2.5 micrograms of the purified rhI2S (the 26thto 550th amino acids of SEQ ID NO: 10) of Lot Nos. 1 to 3, respectively.

FIG. 4 shows the SE-HPLC chart of the purified rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) (Lot No. 1). The vertical and horizontalaxes show absorbance at 215 nm and retention time, respectively.

FIG. 5 shows the SAX-HPLC chart of the purified rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) (Lot No. 1). The vertical and horizontalaxes show absorbance at 280 nm and retention time, respectively.

FIG. 6 shows the result of the measurement of fibroblast uptake of rhI2S(the 26th to 550th amino acids of SEQ ID NO: 10) (Lot No. 1) and acommercially-available medicinal rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10). The vertical axis shows the amount (ng) of rhI2S (the26th to 550th amino acids of SEQ ID NO: 10) taken up per unit mass (mg)of the total cellular protein (ng/mg total cellular protein). Horizontalaxis shows the concentration of rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) in the culture medium. Filled circles and filledtriangles indicate the values of fibroblast uptake of rhI2S (the 26th to550th amino acids of SEQ ID NO: 10) (Lot No. 1) and a commerciallyavailable medicinal rhI2S (the 26th to 550th amino acids of SEQ ID NO:10), respectively. An open circle and a filled triangle close to thehorizontal axis indicate the value of fibroblast uptake of rhI2S (the26th to 550th amino acids of SEQ ID NO: 10) (Lot No. 1) andcommercially-available medicinal rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) in the presence of 20 mmol/L M6P, respectively.

FIG. 7 shows a schematic diagram of the instrument and flow path formeasurement of M6P content. 1: autosampler, 2: column heater, 3: column,4: heat block, 5: water bath, 6: back pressure regulator, 7:fluorescence detector, A: mobile phase A, B: mobile phase B, C: reactionbuffer

DESCRIPTION OF EMBODIMENTS

In the present invention, while recombinant human iduronate 2-sulfatase(rhI2S) (the 26th to 550th amino acids of SEQ ID NO: 10) is preferably arecombinant protein of human wild-type I2S comprising a singleglycosylated polypeptide composed of 525 amino acids, it does notexclude recombinant proteins of human mutant-type I2S's, which have oneor more substitution, deletion, addition, and insertion of one or moreamino acids compared with the amino acid sequence of human wild-typeI2S. The amino acid sequence of human wild-type I2S, including anN-terminal signal sequence, and the DNA sequence encoding it are shownas SEQ ID NO:9 and SEQ ID NO:10, respectively. The N-terminal signalsequence consists of 25 amino acids and is removed post-translationally.

In the present invention, the term “recombinant human I2S-producingmammalian cells” or “rhI2S (the 26th to 550th amino acids of SEQ ID NO:10)-producing mammalian cells” means mammalian cells which have beenartificially manipulated to express, or strongly express, the geneencoding human I2S. Though in general the gene to be strongly expressedis one which is introduced to the mammalian cells (transformation) usingan expression vector in which the gene is incorporated, it may be alsoan intrinsic gene which has been artificially modified in such a mannerthat the gene comes to be strongly expressed. Examples of the means forartificially modifying an intrinsic gene to make it strongly expressitself include, but not limited to, replacement of the promoter upstreamof the intrinsic gene with a promoter which strongly induces expressionof the gene. Such methods have been disclosed in several literatures(e.g., WO94/12650, and WO95/31560). Though there is no particularlimitation as to which mammalian cells are to be employed, preferred arethose of human-, mouse- or hamster-origin, and, among others, CHO cells,which originate from Chinese hamster ovary cells, are particularlypreferred.

In the present invention, the term “recombinant human I2S” or “rhI2S(the 26th to 550th amino acids of SEQ ID NO: 10)” means the human I2Swhich is secreted by the above-mentioned rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10)-producing mammalian cells in the medium duringculture.

In the present invention, the term “oligosaccharide chain” means a chainof oligosaccharide covalently linked to the peptide chain of I2S,including an asparagine type sugar chain, which is covalently bound toan asparagine residue of I2S.

As used in the present invention, the term “fluoroapatite” means amaterial comprising an insoluble fluoridated mineral of calciumphosphate with the chemical formula Cas (PO₄)₃F. Apart from the compoundwhich naturally occurs, fluoroapatite is artificially produced byreplacing the hydroxyl groups of hydroxylapatite (Ca₅(PO₄)₃OH), andproducts in which the hydroxyl groups are almost completely replacedwith fluoride ions are commercially available (e.g., CFT Type II 40micrometer (Bio-Rad Laboratories).

In the present invention, an example of preferable serum-free media inwhich the rhI2S (the 26th to 550th amino acids of SEQ ID NO:10)-producing mammalian cells are to be cultured is the following mediumwhich comprises; 3-700 mg/mL of amino acids, 0.001-50 mg/L of vitamins,0.3-10 g/L of monosaccharides, 0.1-10000 mg/L of inorganic salts,0.001-0.1 mg/L of trace elements, 0.1-50 mg/L of nucleosides, 0.001-10mg/L of fatty acids, 0.01-1 mg/L of biotin, 0.1-20 micrograms/L ofhydrocortisone, 0.1-20 mg/L of insulin, 0.1-10 mg/L of vitamin B₁₂,0.01-1 mg/L of putrescine, 10-500 mg/L of sodium pyruvate, andwater-soluble iron compounds. As desired, it may also include thymidine,hypoxanthine, a conventional pH indicator, and antibiotics.

Further, DMEM/F12 medium, a mixed medium consisting of DMEM and F12, mayalso be used as a basic serum-free medium. Both of these media are wellknown to those skilled in the art. As a serum-free medium, DMEM(HG)HAMmodified (R5) medium may be used, too, which contains sodium hydrogencarbonate, L-glutamine, D-glucose, insulin, sodium selenite,diaminobutane, hydrocortisone, ferric (II) sulfate, asparagine, asparticacid, serine, and polyvinyl alcohol. Furthermore, a commerciallyavailable serum-free medium may also be used as a basic medium.

Each of the chromatography procedures for purification of the rhI2S (the26th to 550th amino acids of SEQ ID NO: 10) may, when needed, be carriedout in the presence of a nonionic surfactant in order to preventnonspecific binding of the protein. Though there is no particularlimitation as to which nonionic surfactant is to be employed, apolysorbate-based surfactant is preferably employed, and more preferablypolysorbate 80 or polysorbate 20. The concentration of such a nonionicsurfactant is preferably 0.005% (w/v) to 0.05% (w/v), more preferably0.01% (w/v).

The process for purification of the rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) may be carried out at room temperature or at lowertemperatures, but preferably at lower temperatures, particularly at 1-10deg C.

In the first chromatography step for the purification process, the rhI2S(the 26th to 550th amino acids of SEQ ID NO: 10) is let bind to thecationic-exchange column that has been equilibrated with an acetatebuffer supplemented with a salt. The pH of this acetate buffer has beenadjusted preferably to 4.0-4.6, more preferably to about 4.2-4.4, andstill more preferably to about 4.3. Though there is no particularlimitation as to which salt is to be added to the phosphate buffer,sodium chloride is preferred, and its concentration is preferably in therange of 50-250 mM, and more preferably in the range of 100-200 mM.

After the column to which rhI2S (the 26th to 550th amino acids of SEQ IDNO: 10) is bound is washed, the rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) is eluted using an acetate buffer with an increased pH,preferably in the range of about 4.8-6.5, more preferably in the rangeof about 5.0-5.4 and still more preferably about 5.1. Salt should becontained in this acetate buffer. Though there is no particularlimitation as to which salt should be contained to the acetate buffer,sodium chloride is preferred, and its concentration is preferably in therange of 50-250 mM, and more preferably in the range of 100-200 mM.

Further, though there is no particular limitation as to whichcation-exchanger is to be employed in the cation-exchange columnchromatography, a weak cation exchanger is preferred, and more preferredis a weak cation exchanger having a selectivity based on bothhydrophobic interaction and hydrogen bond formation. For example, a weakcation exchanger having phenyl groups, amide bonds and carboxyl groupsand having a selectivity based on both hydrophobic interaction andhydrogen bond formation, such as Capto MMC (GE Healthcare), etc., may beemployed.

The dye affinity chromatography, the second step of the purificationprocess, is a step for removing contaminants making use of the strongaffinity of human I2S to certain dyes. Blue triazine dye is preferablyused, but other triazine dyes are also suitable. A particularlypreferred column material for this is Blue Sepharose 6 FF, Fast Flow (GEHealthcare) in which the dye, Cibacron™ Blue F3GA, is covalentlyimmobilized to Sepharose 6 Fast Flow matrix.

The dye affinity chromatography column is equilibrated with an acetatebuffer supplemented with a salt. The pH of this acetate buffer has beenadjusted preferably to 4.5-5.5, more preferably to about 4.8-5.2, andstill more preferably to about 5.0. Though there is no particularlimitation as to which salt is to be added to the phosphate buffer,sodium chloride is preferred, and its concentration is preferably in therange of 50-200 mM, and more preferably in the range of 80-120 mM.

RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10)-containingfractions of the eluate obtained in the first step are diluted with 1-2volumes of water, and adjusted preferably at pH 4.5-5.5, more preferablyat pH 4.8-5.2, and still more preferably at about pH 5.0, then theeluate are applied to the column.

After the column to which rhI2S (the 26th to 550th amino acids of SEQ IDNO: 10) is bound is washed, the rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) is eluted. Elution may be done by an acetate buffer withan increased pH. The concentration of a salt in the buffer may beincreased at the same time. The pH of this acetate buffer has beenadjusted preferably to 5.5-6.5, more preferably to 5.8-6.2, and furtherpreferably to about 6.0. In the case the concentration of a salt is alsoincreased, its concentration is preferably in the range of 100-200 mM,more preferably in the range of 120-170 mM, and still more preferablyabout 150 mM.

The anion-exchange column chromatography, the third step of thepurification process, is a step to eliminate contaminant proteins.Though there is no particular limitation as to which anion-exchangerresin is to be employed, strong anion-exchanger resin may be usedpreferably. Commercially available resins, such as Q Sepharose Fast Flow(GE Healthcare), may be used preferably.

In the anion-exchange column chromatography, the column is equilibratedwith a phosphate buffer supplemented with a salt. The pH of thisphosphate buffer has been adjusted preferably to 5.0-6.0, morepreferably to about 5.2-5.8, and still more preferably to about 5.5.Though there is no particular limitation as to which salt is to be addedto the phosphate buffer, sodium chloride is preferred, and itsconcentration is preferably in the range of 100-200 mM, more preferablyin the range of 120-180 mM, and still more preferably about 150 mM.

pH of the rhI2S (the 26th to 550th amino acids of SEQ ID NO:10)-containing fractions of the eluate obtained in the second step isadjusted preferably to 5.0-6.0, more preferably to pH 5.2-5.8, andfurther preferably to about pH 5.5. The eluate then is applied to thecolumn.

After the anion-exchanger column to which rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) is bound is washed, the rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) is eluted using a phosphate buffer withincreased salt concentration, preferably to 300-500 mM, more preferablyto 350-450 mM, and still more preferably to about 400 mM.

The column chromatography employing a solid phase having affinity tophosphate group, the fourth step of the purification, is a step toeliminate not only contaminant proteins but also such rhI2S (the 26th to550th amino acids of SEQ ID NO: 10) molecules that have aoligosaccharide chain containing comparatively few mannose 6-phosphateresidues. Though there is no particular limitation as to which solidphase is to be used having affinity for phosphate group, fluoroapatiteand hydroxyapatite may preferably be used, and fluoroapatite isparticularly preferred.

Fluoroapatite has affinity for a phosphate group on the oligosaccharidechain. Thus, the more mannose 6-phosphate residues rhI2S (the 26th to550th amino acids of SEQ ID NO: 10) has in its oligosaccharide chain,the more selectively it is bound to fluoroapatite. Though there is noparticular limitation as to which fluoroapatite is to be employed as thesolid phase, commercially available fluoroapatite, such as CFT Type II40 micrometer (Bio-Rad Laboratories), may be used preferably.

The fluoroapatite column is equilibrated with a phosphate buffersupplemented with a salt. The pH of this phosphate buffer has beenadjusted preferably to 6.0-7.0, more preferably to about 6.3-6.7, andstill more preferably to about 6.5, and the concentration of phosphatepreferably to 4.0-15.0 mM, more preferably to about 5.0-10.0 mM, andfurther preferably to about 7.5 mM. Though there is no particularlimitation as to which salt is to be added to the phosphate buffer,sodium chloride is preferred, and its concentration is preferably in therange of 300-500 mM, and more preferably in the range of 350-450 mM.

RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10)-containingfractions of the eluate obtained in the third step are diluted with 2-4volumes of water, and adjusted preferably to pH 6.0-7.0, more preferablyto pH 6.3-6.7, and still more preferably to about pH 6.5, and thisdiluted eluate then are applied to the column.

After the column to which rhI2S (the 26th to 550th amino acids of SEQ IDNO: 10) is bound is washed, the rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) is eluted. Elution may be done by a phosphate buffercontaining a salt. The pH of this phosphate buffer has been adjustedpreferably to 6.0-7.0, more preferably to about 6.3-6.7, and still morepreferably to about 6.5, and the concentration of phosphate is adjustedpreferably to 100 to 200 mM, more preferably to about 125-175 mM, andstill more preferably to about 150 mM. Though there is no particularlimitation as to which salt is to be used, potassium chloride maypreferably be used, at a concentration preferably of 50-250 mM, morepreferably of 100-200 mM, and still more preferably of about 150 mM.

The gel filtration column chromatography, the fifth step of thepurification process, is a step for elimination of low molecular-weightimpurities, such as endotoxins, as well as multimeric complexes ordecomposition products of rhI2S (the 26th to 550th amino acids of SEQ IDNO: 10). Thus, substantially pure rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) is obtained through these consecutive steps, from thefirst to the fifth.

A step for virus inactivation may optionally be added to thepurification process of the present invention. Though there is noparticular limitation as to which process for virus inactivation is tobe applied, solvent-detergent method may preferably be applied. Forthis, nonionic surfactant is added to a solution containing rhI2S (the26th to 550th amino acids of SEQ ID NO: 10), and the mixture isincubated for more than 3 hours. Though there is no particularlimitation as to which surfactant is to be used, polysorbate 20,polysorbate 80, tritonX-100, and tri(n-butyl)phosphate may preferably beused alone or in any combination, and more preferably the combination ofpolysorbate 80 and tri(n-butyl)phosphate may be used.

Such an additional step for virus inactivation may be interposed betweenany two adjacent steps of the purification process mentioned above,particularly between the second and third steps of the purificationprocess (i.e., the step of dye affinity chromatography and that ofanion-exchange column chromatography).

In the present invention, the method for production of rhI2S (the 26thto 550th amino acids of SEQ ID NO: 10) includes at least a step in whichfractions containing rhI2S (the 26th to 550th amino acids of SEQ ID NO:10) is subjected to column chromatography using a solid phase havingaffinity for phosphate group, where, as such a solid phase,fluoroapatite and hydroxyapatite may preferably be used, of whichfluoroapatite is particularly preferred. The step, using a solid phasehaving affinity for phosphate group, is employed for preferentiallypurifying rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10)containing a greater number of mannose 6-phosphate residues in itsoligosaccharide chain. The step can be employed in combination with atleast one other purification step chosen from cation-exchange columnchromatography, dye-affinity column chromatography, anion-exchangecolumn chromatography, and gel filtration column chromatography, in anyorder. A combination is preferred which consists of cation-exchangecolumn chromatography, dye-affinity column chromatography,anion-exchange column chromatography, and then column chromatographyusing a solid phase having affinity for phosphate group, in this order.More preferred is a combination consisting of cation-exchange columnchromatography, dye-affinity column chromatography, anion-exchangecolumn chromatography, column chromatography employing a solid phasehaving affinity for phosphate group, and then gel filtration columnchromatography, in this order.

The present invention provides rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) having an oligosaccharide chain linked thereto whichcontains one or more mannose-6-phosphate residues, wherein the averagenumber of the mannose-6-phosphate residues per rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) molecule is preferably in the range of 3.5to 6.5, more preferably 3.5 to 6.0, still more preferably 3.6 to 5.5,further more preferably 3.7 to 5.4. Alternatively, the average number ofthe mannose-6-phosphate residues per rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) molecule is preferably in the range of 4.0 to6.5, more preferably 4.0 to 6.0, still more preferably 4.2 to 5.8,further more preferably 4.5 to 5.4. The number of themannose-6-phosphate residues per rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) molecule can be determined by the method described below.

The present invention further provides rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10), wherein the average dissociation constantbetween the rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) andmannose-6-phosphate receptor is preferably in the range of 7.0 to25×10⁻¹⁰ mol/L, more preferably 7.5 to 20×10⁻¹⁰ mol/L, still morepreferably 7.5 to 15×10⁻¹⁰ mol/L, and further more preferably 7.5 to13×10⁻¹⁰ mol/L. Alternatively, the average dissociation constant betweenthe rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) andmannose-6-phosphate receptor is preferably in the range of 4.5 to20×10⁻¹⁰ mol/L, more preferably 7.5 to 20×10⁻¹⁰ mol/L, still morepreferably 5.0 to 15×10⁻¹⁰ mol/L, and further more preferably 7.5 to13×10⁻¹⁰ mol/L. The dissociation constant can be determined by themethod described below.

RhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) described abovecan be purified by a purification process described in detail belowincluding at least a step in which fractions containing rhI2S (the 26thto 550th amino acids of SEQ ID NO: 10) is subjected to columnchromatography using a solid phase having affinity for phosphate groups.Preferable examples of such a solid phase include fluoroapatite andhydroxyapatite, of which fluoroapatite is more preferred.

As an rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) molecule isincorporated into its target cells through its binding to the M6Preceptor expressed on the surface of the cells, rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) provided by the present invention can besorted into the target cells more efficiently.

The present invention is described in further detail below withreference to examples. However, it is not intended that the presentinvention be limited to the examples.

Example 1 1. Construction of Human I2S Expression Vector

Vector pEF/myc/nuc (Invitrogen) was digested with KpnI and NcoI to cutout a region including the EF-1(alpha) promoter and its first intron,which then was blunt-ended with T4 DNA polymerase. Vector pC1-neo(Invitrogen) was digested with BglII and XbaI to cut out a regionincluding the CMV enhancer/promoter and the chimeric intron, which thenwas blunt-ended with T4 DNA polymerase. Into this was inserted theabove-mentioned region including the EF-1(alpha) promoter and its firstintron to give pE-neo vector (FIG. 1).

Using a human placenta cDNA library (Takara Bio) as a template, a nestedPCR reaction was performed with two sets of primers, composed of a setof outer primers:

(a) I2S-f: (SEQ ID NO: 1) 5′-ACGCCTATTGCTGCAGGATG-3′, and (b) I2S-r:(SEQ ID NO: 2) 5′-AAACGACCAGCTCTAACTCC-3′for the 1st reaction, and a set of two inner primers:

(c) I2S-f2: (SEQ ID NO: 3) 5′-ATActcgagGCCACCATGCCGCCACCCCGG-3′, and(d) I2S-r2: (SEQ ID NO: 4) 5′-TTCTTATgcggccgcTCAAGGCATCAACAA-3′for the 2nd reaction to amplify the DNA fragment containing human I2ScDNA. The PCR fragment thus amplified was digested with XhoI(corresponding to the part shown with lower case letters in SEQ ID NO:3)and NotI (corresponding to the part shown with lower case letters in SEQID NO:4) and inserted between SalI and NotI sites of vector pE-neo togive vector pE-neo(I2S) (FIG. 1).

The DNA fragment containing poly adenylation signal sequence of humangrowth hormone gene was synthesized by annealing four syntheticoligonucleotides:

(a) hGH-f1: (SEQ ID NO: 5)5'-GGCCGCTCTAGACCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTC CTAATAAA-3′,(b) hGH-r1: (SEQ ID NO: 6)5′-TGATGCAACTTAATTTTATTAGGACAAGGCTGGTGGGCACTGGAGTGGCAACTTCCAGGGCCAGGAGAGGCACTGGGGAGGGGTCACAGGGAT GCCACCCGGGTCTAGAGC-3′,(c) hGH-f2: (SEQ ID NO: 7)5′-ATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATAGCGCAGCACCATGGCCTGAAATAACCTCTGAAAGAGGAACTTGGTTAGG TAC-3′, and (d) hGH-r2:(SEQ ID NO: 8) 5′-CTAACCAAGTTCCTCTTTCAGAGGTTATTTCAGGCCATGGTGCTGCGCTATTATAGAAGGACACCTAGTCAGACAAAA-3′.

The DNA fragment prepared above was inserted between NotI and KpnI sitesof pE-neo(I2S) to give pE-neo/hGHpA(I2S), in which I2S cDNA was locateddownstream of EF-1 promoter and upstream of poly adenylation signalsequence of human growth hormone gene (FIG. 1-1).

Example 2 2. Production of Recombinant Cells for Expression of Human I2S

CHO cells (CHO-K1: purchased from American Type Culture Collection) wastransfected with the above-mentioned expression vector pE-neo/hGHpA(I2S)using Lipofectamine2000 (Invitrogen) according to the following method.Briefly, on the day before transfection, 1×10⁶ CHO-K1 cells were seededin a 3.5-cm culture dish containing 3 mL of D-MEM/F12 medium containing5% FCS (D-MEM/F12/5% FCS), and the cells were cultured overnight at 37deg C in a humidified atmosphere of 5% CO₂ and 95% air. On the followingday, the cells were transfected with 300 microliters of a 1:1 mixturesolution consisting of Lipofectamine 2000 solution diluted 25 times withOpti-MEM I medium (Invitrogen) and a plasmid DNA solutionpE-neo/hGHpA(I2S) diluted with Opti-MEM I medium to 13.2 micrograms/mL,at 37 deg C in a humidified atmosphere of 5% CO₂ and 95% air over night.

After transfection, the medium was replaced with D-MEM/F12/5% FCSsupplemented with 0.6 mg/ml G418, and a selective culture was carriedout at 37 deg C in a humidified atmosphere of 5% CO₂ and 95% air. Cellsthat had grown in the medium for selective culture were subjected toseveral successive rounds of subculture in the medium to giverecombinant cells.

Then, according to the limiting dilution technique, the recombinantcells were seeded on a 96-well plate in such a manner that not more thanone cell might be seeded per well, and the cells were cultured for about10 days to let each of them form a monoclonal colony. The culturesupernatant in the wells where a monoclonal colony was formed weresampled and examined for human I2S activity as described in Example 6below but without desalting procedure, and cell lines which were foundexpressing a high activity for I2S were selected.

For adaptation to serum-free suspension cell culture, the selected celllines were cultured in a commercially available serum-free medium, ISCHO-V-GS medium (Irvine Scientific) supplemented with 8 mM L-glutamine,120 mg/L G148, at 37 deg C in a humidified atmosphere of 5% CO₂ and 95%air until the cells stably grew. The cells then were suspended in ISCHO-V-GS medium (Irvine Scientific) supplemented with 8 mM L-glutamine,100 micromoles/L of hypoxanthine, 16 micromoles/L of thymidine, 120 mg/LG148, and 10% DMSO, and stored as seed cells in liquid nitrogen.

Example 3 3. Culture of Recombinant Cells for Expression of Human I2S

The above seed cells were thawed and diluted to a cell density of 4×10⁵cells/mL and cultured for 3 to 4 days in IS CHO-V-GS medium supplementedwith 8 mM of L-glutamine, 100 micromoles/L of hypoxanthine, 16micromoles/L of thymidine (IS medium), and then diluted to a celldensity of 2×10⁵ cells/mL with IS medium and cultured for 3 to 4 days.The cells were again diluted to a cell density of 2×10⁵ cells/mL andsubjected to expansion culture which was performed by static-culture for4 days at 37 deg C in a humidified atmosphere of 5% CO₂ and 95% air.

The number of the cells was counted, and the cell culture was dilutedwith IS medium to a cell density of 5×10⁵ cells/mL. 10 mL of CDLC(Chemically defined Lipid Concentrate, Invitrogen) was added to 1 L ofthe diluted culture and then the cells were shake-cultured for 3 days.The culture conditions for this were as follows: shaking speed: 20 rpm,pH 7.2, dissolved oxygen: 70%, temperature: 37 deg C. The scale ofculture was escalated until the culture volume had reached 160 L.

Then the number of the cells was counted, and the cells were diluted toa cell density of 2×10⁵ cells/mL with EX-CELL™ 302 serum free-medium (EXmedium, SAFC Bioscience) supplemented with 4 mM of L-glutamine, 100micromoles/L of hypoxanthine, 16 micromoles/L of thymidine. 720 L of thediluted culture was transferred to an incubation tank and cultured for 7days. The culture conditions for this were as follows: agitation speed:about 90 rpm, pH 7.0, dissolved oxygen: 50%, temperature: 37 deg C. 63 Lof EX-CELL™ 302 serum free-medium supplemented with 3.6 mol ofL-glutamine and 10 g of human insulin was added on the 3rd and the 5thdays. Sampling was made every day during the culture, and measurementwas performed for cell number, survival rate, glucose concentration,lactic acid concentration, and amount of expressed human I2S. In thecase where glucose concentration became lower than 9.5 mmol/L, glucosesolution was added so that the concentration reached 19 mmol/L.

The above-mentioned cell culture was repeated 6 times (Lot Nos. 1-6). Ineach culture, viable cell density reached about 1×10⁷ cells/mL or moreon days 6-7 of culture, indicating that high-density cell culture wassuccessfully achieved (FIG. 2). The concentration of rhI2S (the 26th to550th amino acids of SEQ ID NO: 10) in the medium secreted by the cellswas measured by ELISA and compared to the cell density, which revealedthat the concentration increased with a slight time lag (data notshown).

The cell culture was collected and filtered through Millistak+ HC PodFilter grade DOHC, (Millipore), and then through Millistak+ HC gradeA1HC (Millipore), to give a culture supernatant.

Example 4 4. Method for Purification of rhI2S (the 26th to 550th AminoAcids of SEQ ID NO: 10)

To the culture supernatant collected above was added acetic acid toadjust the pH of the culture supernatant to 4.3. A precipitate formed bythis was removed by filtration through Millistak+HC Pod Filter gradeDOHC (Millipore) and then through Opticap XL4 Durapore (Millipore).Culture supernatant thus recovered was loaded on a Capto MMC column(column volume: 6.3 L, bed height: about 20 cm, GE Healthcare), acation-exchange column having a selectivity based both on hydrophobicinteraction and on hydrogen bond formation, which had been equilibratedwith a threefold column volume of 20 mM acetate buffer (pH 4.3)containing 150 mM NaCl. This buffer then was supplied to the column at alinear flow rate of 150 cm/hr to let rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) be adsorbed by the column. Then after the columnwas washed with a fourfold column volume of the same buffer supplied atthe same flow rate, the adsorbed rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) was eluted with a fourfold column volume of 20 mM acetatebuffer (pH 5.1) containing 150 mM NaCl.

Then, the eluate from the above Capto MMC column was diluted 1.5 timeswith water, and the pH of this diluted solution was adjusted to 5.0.This solution then was loaded on a Blue sepharose 6FF column (columnvolume: about 7.1 L, bed height: about 10 cm, GE Healthcare), a dyeaffinity column, which had been equilibrated with a fourfold columnvolume of 20 mM acetate buffer (pH 5.0) containing 100 mM NaCl. Thisbuffer then was supplied to the column at a linear flow rate of 50 cm/hrto let rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) beadsorbed by the column. Then, the column was washed with a fourfoldcolumn volume of the same buffer supplied at the same flow rate, and theadsorbed rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) then waseluted with a fivefold column volume of 20 mM phosphate buffer (pH 6.0)containing 150 mM NaCl.

Then, as a virus inactivation process, tri-n-butyl phosphate (TNBP) andpolysorbate 80 were added to rhI2S (the 26th to 550th amino acids of SEQID NO: 10)-containing fractions of the eluate from the above Bluesepharose 6FF column so that their final concentrations would be0.3%(v/v) and 1%(w/v), respectively, and this mixture solution then wasgently stirred for 3 hours at room temperature. The solution then wasfiltered through Opticap XL4 Durapore (Millipore).

To the above solution, virus-inactivated, was added diluted hydrochloricacid to adjust its pH to 5.5. This solution then was loaded on a QSepharose Fast Flow column (column volume: about 6.3 L, bed height:about 20 cm, GE Healthcare), an anion-exchange column, which had beenequilibrated with a fourfold column volume of 20 mM phosphate buffer (pH5.5) containing 150 mM NaCl. This buffer then was supplied to the columnat a linear flow rate of 150 cm/hr to let rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) be adsorbed by the column. Then, the column waswashed with a fourfold column volume of the same buffer supplied at thesame flow rate, and the adsorbed rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) then was eluted with a fourfold column volume of 20 mMphosphate buffer (pH 5.5) containing 400 mM NaCl.

Then, the eluate from the above Q Sepharose Fast Flow column was dilutedabout 2.7 times with 400 mM NaCl solution, and then the pH of thisdiluted solution was adjusted to 6.5. This solution then was loaded on aCFT Type II 40 micrometer column (column volume: about 3.2 L, bedheight: about 10 cm, Bio-Rad), a fluoroapatite column, which had beenequilibrated with a ninefold column volume of 7.5 mM phosphate buffer(pH 6.5) containing 400 mM NaCl. This buffer then was supplied at alinear flow rate of 150 cm/hr to let rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) be adsorbed by the column. After the column waswashed with a fifteen-fold column volume of the same buffer supplied atthe same flow rate, the adsorbed rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) then was eluted with a fifteen-fold column volume of 150mM phosphate buffer (pH 6.5) containing 150 mM KCl.

Then, the eluate from the above CFT Type II 40 micrometer column wasconcentrated using Biomax™ 30 membrane (Millipore). About 1.2 L of thisconcentrated solution was loaded on a Superdex 200 prep grade column(column volume: about 19 L, bed height: about 64 cm, GE Healthcare)which had been equilibrated with 20 mM phosphate buffer (pH 6.0)containing 137 mM NaCl and 0.02%(w/v) polysorbate 80. The same bufferthen was supplied at a linear flow rate of 14.9 cm/hr, and fractionswhich exhibited peak absorption at 280 nm were collected as fractionscontaining purified rhI2S (the 26th to 550th amino acids of SEQ ID NO:10).

The fractions containing purified rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) were combined and filtered through Planova™ 15N (0.3m² size, Asahi Kasei Medical) and then through Millipak-20 Filter Unit0.22 micrometer to avoid any possible viral contamination in the finalproduct.

The amount of rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10)after each step was quantified using ELISA method described below. Therecovery rate of rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10)of each purification step is shown in Table 1, in which “rhI2S (the 26thto 550th amino acids of SEQ ID NO: 10) recovery rate/step” means theproportion of the amount of recovered rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) to that of loaded rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) in each step, and “rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) recovery rate/total” the proportion of theamount of recovered rhI2S (the 26th to 550th amino acids of SEQ ID NO:10) in each process to the initial amount of rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) subjected to the purification process. Theamount of rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10)subjected to the above purification process was 14435.2 mg, of which9086.3 mg of rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) wasfinally recovered, thus giving the rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) recovery rate/total as high as 62.9%. These resultsshow that the method for purification described above enables to purifyrhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) in very highyield and in a large production scale.

[Table 1]

TABLE 1 Recovery rate of rhI2S in each purification step (Lot No. 1)rhI2S rhI2S rhI2S rhI2S recovery recovery loaded recovered rate/steprate/total Purification process (mg) (mg) (%) (%) Cation-exchange column14435.2 12800.2 88.7 88.7 (Capto MMC) Dye affinity column 13436.0 9155.468.1 63.4 (Blue Sepharose 6FF) Anion-exchange column 12885.5 12356.595.9 85.6 (Q Sepharose FF) Fluoroapatite column 11719.9 7882.3 67.3 54.6(CFT TypeII) Gel filtration column 8905.5 9086.3 102.0 62.9 (Superdex200 pg)

Example 5 5. Analysis of Human I2S by ELISA

To each well of a 96-well microtiter plate (Nunc) was added 100microliters of mouse anti-human monoclonal antibody diluted to 4micrograms/mL with 0.05 M Carbonate-Bicarbonate buffer (pH 9.6), and theplate was let stand for at least 1 hour at room temperature to let theantibody be absorbed by the wells. Then, after each well was washedthree times with phosphate buffered saline (pH 7.4) containing 0.05%Tween 20 (PBS-T), 200 microliters of Starting Block (PBS) BlockingBuffer (Thermo Fisher Scientific) was added to the well, and the platewas let stand for at least 30 minutes at room temperature. Then, aftereach well was washed three times with PBS-T, 100 microliters of the testsample or human I2S standard, which had been diluted as desired with PBScontaining 0.5% BSA and 0.05% Tween 20 (PBS-BT), was added to the well,and the plate was let stand for at least one hour at room temperature.Then, after each well was washed three times with PBS-T, 100 microlitersof biotin-labeled anti-human I2S monoclonal antibody diluted with PBS-BTwas added and the plate was let stand for at least 1 hour. Then, aftereach well was washed three times with PBS-T, 100 microliters ofstreptavidin-HRP (R&D SYSTEMS) diluted with PBS-BT was added and theplate was let stand for at least 30 minutes. Then, after each well waswashed three times with PBS-T, 100 microliters of 0.4 mg/mLo-phenylendiamine with phosphate-citrate buffer (pH 5.0) was added tothe well, and the plate was stand for 8 to 20 minutes at roomtemperature. Then 0.1 mL of 1 mol/L H₂SO₄ was added to each well to stopthe reaction, and the optical density at 490 nm was measured for thewell on a 96-well plate reader.

Example 6 6. Measurement of the Activity of rhI2S (the 26th to 550thAmino Acids of SEQ ID NO: 10)

Samples were desalted by membrane filtration using verticalpolyethersulfone membrane (VIVASPIN 2 5,000 MWCO PES; Sartorius) asultrafilter membrane, and then desalted samples were diluted toapproximately 100 ng/mL with Reaction Buffer (5 mM sodium acetate, 0.5mg/L BSA, 0.1% Triton X-100, pH 4.45). To each well of a 96-wellmicrotiter plate (FluoroNunc Plate, Nunc) 10 microliters of each rhI2S(the 26th to 550th amino acids of SEQ ID NO: 10) sample was added andpre-incubated for 15 minutes at 37 deg C. Substrate solution wasprepared by dissolving 4-methyl-umbelliferyl sulfate (SIGMA) inSubstrate Buffer (5 mM sodium acetate, 0.5 mg/mL BSA, pH 4.45) to afinal concentration of 1.5 mg/mL. 100 microliters of Substrate solutionwas added to each well containing rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) sample and the plate was let stand for 1 hour at 37deg. C in the dark. After the incubation, 190 microliters of Stop Buffer(0.33 M glycine, 0.21 M sodium carbonate, pH 10.7) was added to eachwell containing the sample. 150 microliters of 0.4 micromole/L4-methylumbelliferone (4-MUF, Sigma) solution and 150 microliters ofStop Buffer was added to a well as the standard, then the plate was readon a 96-well plate reader with excitation light at the wavelength of 330nm and fluorescent light at the wavelength of 440 nm.

A standard curve was produced by measuring fluorescence intensity atvarious concentrations of 4-MUF in solution. The fluorescence intensityof each sample was extrapolated to the standard curve. Results werecalculated as activity in Units/mL where one Unit of activity was equalto 1 micromole of 4-MUF produced per minute at 37 deg. C. A published USpatent application (publication No. 2004-0229250) was referred to forconducting this measurement. The specific activity of human I2S purifiedabove was found to be about 2630 mU/mg.

Example 7 7. Measurement of Host Cell Proteins

A rabbit was immunized with proteins obtained from CHO cells (host cellproteins: HCPs), then antiserum was prepared from blood of the rabbit.Total IgG was purified from the antiserum by protein A columnchromatography, then the antibody against HCPs (rabbit anti-HCPantibody) was purified from the total IgG by affinity columnchromatography containing HCP-coupled resin. Biotin-labeled rabbitanti-HCP antibody was prepared by conjugating rabbit anti-HCP antibodyto biotin moiety using EZ-Link Sulfo-NHS-LC-Biotinylation Kit (Thermo).

One hundred microliters of Rabbit anti-HCP antibody diluted to 5micrograms/mL with 100 mM sodium carbonate buffer (pH 9.6) was added toeach well of a 96-well microtiter plate (Nunc). After the plate was letstand for at least 1 hour at room temperature to allow the antibody tobe adsorbed, each well was washed three times with T-TBS (Tris bufferedsaline with 0.05% Tween 20, pH 8.0, SIGMA), then 200 microliters ofSuper Block Blocking Buffer in TBS (Thermo) was added to each well, andthe plate was let stand for at least 30 minutes at room temperature.Then each well was washed three times with T-TBS, then 100 microlitersof sample solution diluted with Super Block Blocking Buffer in TBS(Thermo) was added, and the plate was rotated mildly for at least 1 hourat room temperature. After each well was washed three times with T-TBS,100 microliters of 60 ng/mL biotin-labeled rabbit anti-HCP antibody inSuper Block Blocking Buffer in TBS (Thermo) was added, and the plate wasrotated mildly for at least 1 hour at room temperature. Then each wellwas washed three times with T-TBS, and following addition of 100microliters of horseradish peroxidase-labeled streptavidin (NeutrAvidin,Horseradish Peroxidase Conjugated, Thermo) diluted with Super BlockBlocking Buffer in TBS (Thermo), the plate was rotated mildly for atleast 30 minutes at room temperature. Each well then was washed threetimes with T-TBS, and after addition of 100 microliters of TMB solution(TMB Peroxidase Substrate, KPL), the plate was let stand for 15 to 30minutes at room temperature. To each well was added 100 microliters of 1M phosphoric acid to stop the reaction, and the optical density at 450nm was measured for each well on a 96-well plate reader. As a result,the calculated concentration of HCPs in the rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) purified above (Lot No. 1) was only 12ppm, which is the level acceptable for using this rhI2S (the 26th to550th amino acids of SEQ ID NO: 10) as a medicament injectable to humanbody.

Example 8 8. Analysis of Purity of rhI2S (the 26th to 550th Amino Acidsof SEQ ID NO: 10)

The rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) purifiedabove was subjected to SDS-PAGE electrophoresis under non-reductive,heating condition. The gel-stained with Coomassie brilliant bluerevealed a single band at the position of molecular weight of about 80kD, which corresponds to the molecular weight of rhI2S (the 26th to550th amino acids of SEQ ID NO: 10) reported previously (U.S. Pat. No.5,932,211) (FIG. 3).

Further, rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) purifiedabove was analyzed by Size-elimination HPLC (SE-HPLC) and SAX-HPLC. HPLCwas performed using LC-20A System, SPD-20AV UV/VIS Detector (ShimazuCorp.).

For SE-HPLC analysis, 10 microliters of sample solution containing 2mg/mL of rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) purifiedabove was applied onto TSK gel G3000SW_(XL) column (7.8 mm I.D.×30 cm,TOSOH) equilibrated with 25 mM phosphate buffered saline (PBS) at a flowrate of 0.5 ml/min. Elution profile was produced by monitoringabsorbance at 215 nm. For SAX-HPLC analysis, 20 microliters of a samplesolution containing 2 mg/mL of rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) was applied onto TSK gel Q-STAT column (4.6 mm I.D.×10cm, TOSOH) equilibrated with 10 mM Tris-HCl (pH 7.5) at a flow rate of1.0 ml/min. NaCl concentration in this mobile phase buffer was increasedup to 0.5 M at 30 min after injection with linear slope. Elution profilewas produced by monitoring absorbance at 280 nm.

Both of SE-HPLC and SAX-HPLC of the above purified rhI2S (the 26th to550th amino acids of SEQ ID NO: 10) revealed a single peak alone (FIGS.4 and 5, respectively). These data showed that the rhI2S (the 26th to550th amino acids of SEQ ID NO: 10) obtained above was highly purifiedand free of any detectable contaminants. Furthermore, the concentrationof host cell proteins (HCPs) contaminating the purified rhI2S (the 26thto 550th amino acids of SEQ ID NO: 10) was only 12 ppm as measured byELISA. Taken together, the data shown above demonstrate that the rhI2S(the 26th to 550th amino acids of SEQ ID NO: 10) purified above is sucha high purity as may be directly used as a medical drug.

Example 9 9. Measurement of Cellular Uptake of rhI2S (the 26th to 550thAmino Acids of SEQ ID NO: 10) Using Normal Human Fibroblast

Cultured human fibroblasts (CCD-1076SK, purchased from DS PharmaBiomedical Co., Ltd.) were suspended in MEM-Eagle's medium (Gibco)containing 10% heat-inactivated FBS and 2 mM L-glutamine, and the celldensity was adjusted to 8.0×10⁴ cells/mL. One hundred microliters ofcell suspension was seeded into each well of a 96-well microplate andcultured for 2 days at 37 deg C in a humidified atmosphere of 5% CO₂ and95% air. Samples containing the present rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) obtained above (Lot No. 1) or acommercially-available medicinal rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) were serially diluted with the medium to make variousconcentrations of from 4.88 ng/mL to 20 micrograms/mL. Then, the mediumin the 96-well microplate where fibroblasts had been seeded was removedwith a micropipette, and 100 microliters of each sample diluted abovewas added in duplicate manner to the wells, and incubation was done for18 hrs. The final concentrations of rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) in the incubation was 4.88 ng/mL to 20 micrograms/mL.To confirm the specific binding of rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) to mannose 6-phosphate (M6P) receptor in this assay,samples containing 10 mmol/L of mannose 6-phosphate (M6P) as anantagonist and 20 micrograms/mL of rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) were prepared and added to the 96-well microplate, andincubated was done in the same manner as described above. The assay wasmade in triplicated manner.

After washed with ice cold PBS thrice, cells were lysed with M-PERMammalian Protein Extraction Reagent (Thermo scientific) supplementedwith 0.5% protease inhibitor cocktail (Sigma). Then the amount of totalcellular protein was measured by Pierce BCA™ Protein Assay kit (Pierce,Ill., USA), and the amount of rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) taken up in the cells was determined by ELISA method asdescribed above. The amount of rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) taken up was calculated per unit mass (mg) of the amountof total cellular protein, and plotted on a graph (FIG. 6).

The result showed that the present rhI2S (the 26th to 550th amino acidsof SEQ ID NO: 10) (Lot No. 1) was taken up by cultured human fibroblastsin a dose-dependent manner more efficiently than thecommercially-available medicinal rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) (FIG. 6). The EC₅₀ of cellular uptake of the presentrhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) (Lot No. 1) andthe commercially-available medicinal rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) was 0.452+/−0.0853 and 1.455+/−0.434micrograms/mL (Mean+/−s.d.), respectively. Addition of M6P to theculture at the concentration of 10 mmol/L inhibited the cellular uptakeof both of the present rhI2S (the 26th to 550th amino acids of SEQ IDNO: 10) (Lot No. 1) and the commercially-available medicinal rhI2S (the26th to 550th amino acids of SEQ ID NO: 10) almost completely,indicating that they were taken up by cultured human fibroblasts throughits specific binding to the M6P receptor. Non-patent document (TsukimuraT. et. al., Biol Pharm Bull. 31: 1691-5, 1979) was referred to forconducting the above measurement. These results show that both of thepresent rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) (LotNo. 1) and the commercially-available medicinal rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) contain one or more M6P residues in itsoligosaccharide chain.

Example 10 10. Measurement of Affinity of rhI2S (the 26th to 550th AminoAcids of SEQ ID NO: 10) to M6P Receptor

Binding affinity of rhI2S (the 26th to 550th amino acids of SEQ ID NO:10) for the M6P receptor was measured by the method described below.

Plasmid containing cDNA encoding human mannose-6-phosphate receptor(human M6P receptor), which is essential for M6P binding, was obtainedfrom ATCC (ATCC No. 95660). A DNA fragment encoding the domain 9 ofhuman M6P receptor (hMPR9) was amplified from the plasmid by PCR using aset of primers:

(a) hMPR9-f: (SEQ ID NO: 11) 5′-ATAATCCATGGTTGTCAGAGTGGAAGGGGAC-3′, and(b) hMPR9-r: (SEQ ID NO: 12) 5′-GCAATGCGGCCGCGAAAGGTGGGCAGGCATAC-3′.

The amplified DNA fragment was digested with NcoI and NotI, and insertedinto the NcoI-NotI site of pET26 vector (Novagen). The resultant plasmidwas designated pET26-MPR9. Two-step PCR reaction was conducted using twosets of primers, which resulted in the amplification of a DNA fragmentshown as SEQ ID NO:13, which encoded hMPR9 containing additionalsequences at both of its 5′- and 3′-termini. As this amplified DNAfragment encodes hMPR9 having Bip signal at its N-terminus and His-tagat its C-terminus, it was designated Bip-tagged-hMPR9.

The first reaction of the 2-step PCR reaction above was conducted usingpET26-MPR9 as template and a set of primers:

(c) MPR9-f2: (SEQ ID SEQ ID NO: 14)5′-GTTGGCCTCTCGCTCGGGAGCGCTGTTGTCAGAGTGGAAGG GGAC-3′, and (d) MPR9-r2:(SEQ ID NO: 15) 5′-ATAATGCGGCCGCTCAGTGATGGTGATGGTGATGTGGCGCGCCGGATCCGAAAGGTGGGCAGGCATAC-3′.Subsequently, using the amplified DNA fragment as template, the secondPCR was conducted using a set of primers:

(e) MPR9-f3: (SEQ ID NO: 16)5′-ATAATCCATGGGATATCTAATAAATATGAAGTTATGCATATTACTGGCCGTCGTCGCCTTTGTTGGCCTCTCG-3′, and (f) MPR9-r3: (SEQ ID NO: 17)5′-ATAATGCGGCCGCTCAGTGATGGTGATGGTGATGTGGCGCGCCGGATCCGAAAGGTGGGCAGGCATAC-3′.Then the resultant DNA fragment was digested with EcoRV and NotI, andligated into the Eco47III-NotI site of the pIB/V5-His-DEST vector(Invitrogen). The resultant plasmid was designated pXBi-MPR9, with whichHigh Five cells were transfected to obtain cells expressingHis-tagged-hMPR9 which was derived from Bip-tagged hMPR9 through removalof Bip signal sequence.

High Five cells (Invitrogen) were grown in 24-well plate until 50%confluent using Express Five medium (Invitrogen) and transfected withthe pXBi-MPR9 using the Hily Max transfection reagent (Dojin chemical,Japan). The cells were cultured in the presence of 30 micrograms/mLblasticidin to select stable transfectant. Stably transfected cells thenwere expanded and cultured in the Erlenmeyer flask (100 mL) for 4 days.The culture then was harvested and centrifuged at 3,000 rpm for 30 min,and the supernatant was collected. The supernatant was filtrated though0.22 micrometer filter (Millapore), then diluted 5-fold withequilibration buffer (10 mM phosphate buffer containing 300 mM NaCl (pH7.2)). The diluted supernatant was applied to the chromatography columnwith Profinity IMAC Ni-charged Resin (bed volume: 1 mL, Bio-Rad)equilibrated with an equilibration buffer, then the column was washedwith 5 bed volumes of the equilibration buffer. Then theHis-tagged-hMPR9 bound to the resin was eluted by applying to the column5 bed volumes of 10 mM NaPO4, 300 mM NaCl and 10 mM imidazole (pH 7.2),and subsequently 5 bed volumes of 10 mM NaPO4, 300 mM NaCl and 300 mMimidazole (pH 7.2). Fractions containing His-tagged-hMPR9 were collectedand concentrated by Amicon 3K (Millipore) with the buffer exchanged to20 mM Tris buffer containing 150 mM NaCl (pH 7.4). The concentration ofthe His-tagged-hMPR9 was determined by measuring absorbance at 280 nm.

Binding affinity of rhI2S (the 26th to 550th amino acids of SEQ ID NO:10) for M6PR was measured using Biacore T100 (GE Healthcare) equippedwith a nitrilotriacetic acid-fixed sensor chip (Series S Sensor Chip NTA“BR-1005-32”). Biacore T100 is a measuring apparatus based on surfaceplasmon resonance (SPR), where a sample containing a ligand is sent at aconstant flow rate onto the surface of the sensor chip on which receptoris fixed. If the ligand binds to the receptor, the mass of the surfacesensor chip is increased due to the mass of the ligand bound to thereceptor, and a shift of the SPR signal can be detected as a change inthe resonance unit (RU) in proportion to the amount of bound ligand. Ingeneral, for proteins, 1 RU is approximately 1 pg/mm². To activate thesensor chip, 10 mM HEPES (pH 7.4) containing 500 micromoles/L NiCl₂, 150mM NaCl, 50 micromoles/L EDTA and 0.05% Surfactant P20 was loaded at theflow rate of 10 microliters/min for 60 sec. Then, approximately 50-100RU of the His-tagged-hMPR9 purified above was applied, and subsequently10 mM HEPES (pH 7.4) containing 150 mM NaCl, 50 micromoles/L EDTA and0.05% Surfactant P20 was loaded at the flow rate of 10 microliters/minfor 60 sec to fix the tagged-hMPR9 on the activated sensor chip. Eachsample was diluted to the concentrations of rhI2S (the 26th to 550thamino acids of SEQ ID NO: 10) of 12.5, 6.25, 3.125 and 1.5625 nmol/Lwith 10 mM HEPES (pH 7.4) containing 150 mM NaCl, 50 micromoles/L EDTAand 0.05% Surfactant P20, and each of the dilutions prepared was appliedat a flow rate of 50 microliters/min for 300 sec to let rhI2S (the 26thto 550th amino acids of SEQ ID NO: 10) bind to the His-tagged-hMPR9 onthe sensor chip. Then, 10 mM HEPES (pH 7.4) containing 150 mM NaCl, 50micromoles/L EDTA and 0.05% Surfactant P20 was run at a flow rate of 50microliters/min for 180 sec while constantly monitoring the dissociationstatus between the His-tagged-hMPR9 and rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10). Subsequently 10 mM HEPES (pH 8.3) containing150 mM NaCl, 350 mM EDTA, and 0.05% Surfactant P20 was run at a flowrate of 50 microliters/min for 60 sec to regenerate the sensor chip. Thedissociation constant (Kd) was automatically calculated from thedissociation status monitored above by Biacore T100 Evaluation Software.

The Kd value (dissociation constant) for the present rhI2S (the 26th to550th amino acids of SEQ ID NO: 10) obtained by the above method (LotNo. 1 to 7) ranged from 7.58 to 12.29×10⁻¹⁰ mol/L, which averaged to10.4×10⁻¹⁰ mol/L, whereas the Kd value for the commercially-availablemedicinal rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) was33.2×10⁻¹⁰ mol/L (See Table 2). The result showed that the present rhI2S(the 26th to 550th amino acids of SEQ ID NO: 10) obtained by the abovemethod bound to the M6P receptor more efficiently than thecommercially-available medicinal rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10), and suggested that highly efficient cellular uptake ofthe present rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) (LotNo. 1) shown in FIG. 6 was caused by the stronger affinity of thepresent rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) for theM6P receptor.

TABLE 2 Kd values and mannose-6-phosphate content rhI2S of presentinvention Commercially Average available Lot. Nos. 1-7 (Lot Nos. 1-7)rhI2S Kd 7.58-12.29 10.4 33.2 [×10⁻¹⁰ mol/L] M6P content 3.74-5.38  4.373.39 [mol/mol]

Example 11 11. Measurement of M6P Content

Number of M6P residue in oligosaccharide chain of rhI2S (the 26th to550th amino acids of SEQ ID NO: 10) was measured by the method describedbelow. Standard solution was prepared by dissolvingD-mannose-6-phosphate, D(+)-mannose, L(−)-fucose, and D(+)-galactosewith water at the concentration of 0.1 mg/mL, 0.1 mg/mL, 0.036 mg/mL and0.1 mg/mL, respectively. Mobile phase A was prepared by dissolving 6.2 gof boric acid in water, adjusting the pH to 9.0 with 2M NaOH, and thenadding water to make a total volume of 1000 mL. Mobile phase B wasprepared by dissolving 6.2 g of boric acid and 11.7 g of NaCl in water,adjusting pH to 9.0 with 2M NaOH, and then adding water to make a totalvolume of 1000 mL. Reaction buffer was prepared by dissolving 10 g ofL-arginine and 30 g of boric acid in water to a total volume of 1000 mL.

A sample containing rhI2S (the 26th to 550th amino acids of SEQ ID NO:10) was desalted by ultrafiltration, and its absorbance at 280 nm wasadjusted with water to 0.4 to 0.6. Then 0.2 mL of this desalted samplewas dried up under reduced pressure, and then dissolved completely in0.1 mL of trifluoroacetic acid. The dissolved sample was heated at 100deg C for 2 hours, then cooled to room temperature, and dried up underreduced pressure. This dried-up sample was dissolved completely inmobile phase A.

Anion exchange chromatography column (Shim-pack ISA-07/52504 (4.0 mmI.D.×250 mm, SHIMAZU) (resin: styrene-divinylbenzene polymer, stationaryphase: quaternary ammonium) was connected to SHIMAZU HPLC System LC-Avp(HPLC system for analysis of reducing sugar). The column was set in acolumn heater (Shim-pack guard column ISA, SHIMAZU) to heat the columnat 65 deg C. The flow path from the outlet of the column was connectedto a heat block (ALB-221, AGC Techno Glass Co., Ltd.) which was adjustedat 150 deg C. The flow path from the heat block passed through a waterbath, and then was connected to a back pressure regulator (U-607, MSInstruments Inc.). Further, the flow path from the back pressureregulator was connected to a fluorescence detector, where the flow wasirradiated with excitation light (wavelength: 320 nm) and thenfluorescence (wavelength: 430 nm) emitted from the flow was detected. Aschematic diagram illustrating the instruments and flow path is given inFIG. 7.

Containers for mobile phase A and mobile phase B were connected to theautosampler of HPLC system and a container for Reaction buffer wasconnected to the flow path so that Reaction buffer was supplied betweenthe outlet of the column and the heat block, as illustrated in FIG. 7.

The column was equilibrated with an initial mobile phase (mixture ofphase A and mobile phase B at a ration of 40% and 60%, respectively).Twenty microliters of the sample solution or the standard solution wasapplied to the equilibrated column, and the initial mobile phase wasapplied for 60 minutes at the flow rate of 0.3 mL/minute. Subsequently,the mobile phase was applied for further 10 minutes at the flow rate of0.3 mL/minute while, linearly increasing the proportion of mobile phaseB from 60% to 100%. The Reaction buffer was constantly applied at theflow rate of 0.2 mL/minute.

The area of the peak detected by the fluorescence detector wascalculated, and the amount of mannose-6-phosphate in the sample wasdetermined by comparing the area of the peak obtained, between thesample and the standard solution.

The number of mannose-6-phosphate residues contained per rhI2S (the 26thto 550th amino acids of SEQ ID NO: 10) molecule was calculated by thefollowing formula, where the numbers 282.12 and 77,000 correspond to themolecular weight of mannose-6-phosphate and the approximate molecularweight of rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10)(including linked oligosaccharide chain), respectively.

The number of mannose-6-phosphate per rhI2S (the 26th to 550th aminoacids of SEQ ID NO: 10) molecule (mol/mol))=amount ofmannose-6-phosphate in the sample (mg)/amount of rhI2S (the 26th to550th amino acids of SEQ ID NO: 10) (mg)×282.12/77,000.

The number of M6P residues per molecule of the present rhI2S (the 26thto 550th amino acids of SEQ ID NO: 10) (Lot Nos. 1 to 7) was analyzed tobe in the range of 3.74 to 5.38 mol/mol, which averaged to 4.37 mol/mol,whereas the number of M6P residue per molecule of thecommercially-available medicinal rhI2S (the 26th to 550th amino acids ofSEQ ID NO: 10) was 3.39 mol/mol (See Table 2). The result shows that thepresent rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) obtainedabove method contains a greater number of M6P residues in theoligosaccharide chain linked to it than the commercially-availablemedicinal rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10), andsuggests that this higher M6P content caused the characteristics of thepresent rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10),including its highly efficient cellular uptake and strong affinity forthe M6P receptor.

As the present rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10)have the properties describe above, including highly efficient cellularuptake, it can be more effectively used than the conventional medicamentin the enzyme replacement therapy of patients suffering from diseasescaused by deficiency of the gene encoding I2S, such as Hunter'ssyndrome. As it is incorporated efficiently into its target cells, thepresent rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10), whenused as a medicinal enzyme in the enzyme replacement therapy, could leadto substantial reduction of the amount of the enzyme to be injected intoa patient. This aspect is very advantageous not only to relatingpatients and medical facilities but also to the financial aspect of themedical service system, because reduction in the required amount ofenzyme in each treatment would eventually result in reduction of therequired total production scale of the enzyme, therefore the total costof its production.

INDUSTRIAL APPLICABILITY

The present invention is utilized for production of recombinant humaniduronate 2-sulfatase (rhI2S) (the 26th to 550th amino acids of SEQ IDNO: 10) in a large scale, with a high purity sufficient for itsadministration to a patient as a medicament, and with attachedoligosaccharide chains containing mannose 6-phosphate residues which isnecessary for rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10) tobe employed in the enzyme replacement therapy for Hunter's syndrome.

[Sequence Listing Free Text]

SEQ ID NO:1=Primer I2S-f

SEQ ID NO:2=Primer I2S-r

SEQ ID NO:3=Primer I2S-f2

SEQ ID NO:4=Primer I2S-r2

SEQ ID NO:5=Synthetic oligonucleotide hGH-f1

SEQ ID NO:6=Synthetic oligonucleotide hGH-r1

SEQ ID NO:7=Synthetic oligonucleotide hGH-f2

SEQ ID NO:8=Synthetic oligonucleotide hGH-r2

SEQ ID NO:9=Human wild-type I2S Amino acid sequence

SEQ ID NO:10=DNA sequence encoding human wild-type I2S

SEQ ID NO:11=Primer hMPR9-f

SEQ ID NO:12=Primer hMPR9-r

SEQ ID NO:13=Synthetic DNA sequence encoding hMPR9 having Bip signal atits N-terminus and His-tag at the C-terminus

SEQ ID NO:14=Primer hMPR9-f2

SEQ ID NO:15=Primer hMPR9-r2

SEQ ID NO:16=Primer hMPR9-f3

SEQ ID NO:17=Primer hMPR9-r3

[Sequence Listing]

GP147-PCT_ST25

The invention claimed is:
 1. A method for producing recombinant humanwild-type iduronate 2-sulfatase (rhI2S) consisting of the 26^(th) to550^(th) amino acids of the amino acid sequence set forth in SEQ IDNO:10, wherein the method comprises: (a) culturing rhI2S-producingmammalian cells in a serum-free medium so as to allow the cells tosecrete the rhI2S in the medium, wherein the cells are those that havebeen transformed to express the gene encoding the rhI2S by introductionof the gene using an expression vector in which the gene isincorporated, (b) collecting culture supernatant by removing the cellsfrom the culture that is obtained in step (a) above, (c) subjecting theculture supernatant collected in step (b) above to cation-exchangecolumn chromatography to collect rhI2S-active fractions, (d) subjectingthe fractions collected in step (c) above to dye affinity columnchromatography to collect rhI2S-active fractions, (e) subjecting thefractions collected in step (d) above to anion-exchange columnchromatography to collect rhI2S-active fractions, (f) subjecting thefractions collected in step (e) above to a column chromatographyemploying as a solid phase, a material having affinity for phosphategroup to collect rhI2S-active fractions, and (g) subjecting thefractions collected in step (1) above to gel filtration columnchromatography to collect rhI2S-active fractions, in the respectiveorder.
 2. The method according to claim 1, wherein the cation exchangeremployed in the cation-exchange column chromatography is a weak cationexchanger.
 3. The method according to claim 2, wherein the weak cationexchanger has phenyl groups, amide bonds and carboxyl groups.
 4. Themethod according to claim 1, wherein the dye employed in the dyeaffinity column chromatography is a blue triazine dye.
 5. The methodaccording to claim 1, wherein the material having affinity for phosphategroup is selected from the group consisting of fluoroapatite andhydroxyapatite.
 6. The method according to claim 5, wherein the materialhaving affinity to phosphate group is fluoroapatite.
 7. The methodaccording to claim 1, wherein the mammalian cells are CHO cellstransfected with an expression vector which is designed to express rhI2Sunder the regulation of EF-1(alpha) promoter.
 8. A method for purifyingrecombinant human wild-type iduronate 2-sulfatase (rhI2S) consisting ofthe 26^(th) to 550^(th) amino acids of the amino acid sequence set forthin SEQ ID NO:10 from contaminants in a sample, wherein the rhI2S has anoligosaccharide chain linked thereto containing one or more mannose6-phosphate residues, the method comprising: (a) applying the sample toa chromatography column which employs fluoroapatite as a solid phase,(b) flowing a first mobile phase through the column to wash the columnwhile letting the rhI2S be adsorbed by the column, and (c) eluting therhI2S from the column by flowing a second mobile phase through thecolumn, wherein the second mobile phase contains a phosphate at a higherconcentration than the first mobile phase.